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Surapol Issaragrisil - One of the best experts on this subject based on the ideXlab platform.

  • one step genetic correction of Hemoglobin e beta Thalassemia patient derived ipscs by the crispr cas9 system
    Stem Cell Research & Therapy, 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak Upratya, Surapol Issaragrisil
    Abstract:

    Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future.

  • Additional file 2: Figure S1. of One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Showing characterization of iPSCs derived from skin fibroblasts of a patient with Hemoglobin E/beta-Thalassemia. (a) Pluripotent gene expression of wild-type human dermal fibroblasts (wt-HDFs), parental human dermal fibroblasts (Eβ-HDFs) and Eβ-iPSCs compared with hESC line Chula2.hES, analyzed by RT-PCR. (b) Immunofluorescent staining shows expression of pluripotent markers NANOG, OCT4, SSEA-4, TRA-1-60 and TRA-1-81 in the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars = 100 μm. (c) Immunofluorescent staining shows expression of lineage markers NESTIN (ectoderm), AFP (endoderm) and SMA (mesoderm) of differentiated embryoid bodies generated from the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars: for NESTIN and SMA = 100 μm; for AFP = 50 μm. (d) Hematoxylin and eosin (H&E) staining of teratomas derived from the Eβ-iPSC2 cells at 8 weeks post implantation into nude mice. Teratomas contained tissues derived from three embryonic germ layers, sebaceous tissue (ectoderm), cartilage (mesoderm) and gut-like epithelium (endoderm). Scale bars = 100 μm. (e) Representative karyotypic analysis of the Eβ-iPSC2 cells at passage 19 shows normal karyotype (46, XY) (TIFF 9760 kb

  • One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    'Springer Science and Business Media LLC', 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. Results The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future

Methichit Wattanapanitch - One of the best experts on this subject based on the ideXlab platform.

  • one step genetic correction of Hemoglobin e beta Thalassemia patient derived ipscs by the crispr cas9 system
    Stem Cell Research & Therapy, 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak Upratya, Surapol Issaragrisil
    Abstract:

    Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future.

  • Additional file 2: Figure S1. of One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Showing characterization of iPSCs derived from skin fibroblasts of a patient with Hemoglobin E/beta-Thalassemia. (a) Pluripotent gene expression of wild-type human dermal fibroblasts (wt-HDFs), parental human dermal fibroblasts (Eβ-HDFs) and Eβ-iPSCs compared with hESC line Chula2.hES, analyzed by RT-PCR. (b) Immunofluorescent staining shows expression of pluripotent markers NANOG, OCT4, SSEA-4, TRA-1-60 and TRA-1-81 in the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars = 100 μm. (c) Immunofluorescent staining shows expression of lineage markers NESTIN (ectoderm), AFP (endoderm) and SMA (mesoderm) of differentiated embryoid bodies generated from the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars: for NESTIN and SMA = 100 μm; for AFP = 50 μm. (d) Hematoxylin and eosin (H&E) staining of teratomas derived from the Eβ-iPSC2 cells at 8 weeks post implantation into nude mice. Teratomas contained tissues derived from three embryonic germ layers, sebaceous tissue (ectoderm), cartilage (mesoderm) and gut-like epithelium (endoderm). Scale bars = 100 μm. (e) Representative karyotypic analysis of the Eβ-iPSC2 cells at passage 19 shows normal karyotype (46, XY) (TIFF 9760 kb

  • One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    'Springer Science and Business Media LLC', 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. Results The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future

Nattaya Damkham - One of the best experts on this subject based on the ideXlab platform.

  • one step genetic correction of Hemoglobin e beta Thalassemia patient derived ipscs by the crispr cas9 system
    Stem Cell Research & Therapy, 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak Upratya, Surapol Issaragrisil
    Abstract:

    Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future.

  • Additional file 2: Figure S1. of One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Showing characterization of iPSCs derived from skin fibroblasts of a patient with Hemoglobin E/beta-Thalassemia. (a) Pluripotent gene expression of wild-type human dermal fibroblasts (wt-HDFs), parental human dermal fibroblasts (Eβ-HDFs) and Eβ-iPSCs compared with hESC line Chula2.hES, analyzed by RT-PCR. (b) Immunofluorescent staining shows expression of pluripotent markers NANOG, OCT4, SSEA-4, TRA-1-60 and TRA-1-81 in the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars = 100 μm. (c) Immunofluorescent staining shows expression of lineage markers NESTIN (ectoderm), AFP (endoderm) and SMA (mesoderm) of differentiated embryoid bodies generated from the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars: for NESTIN and SMA = 100 μm; for AFP = 50 μm. (d) Hematoxylin and eosin (H&E) staining of teratomas derived from the Eβ-iPSC2 cells at 8 weeks post implantation into nude mice. Teratomas contained tissues derived from three embryonic germ layers, sebaceous tissue (ectoderm), cartilage (mesoderm) and gut-like epithelium (endoderm). Scale bars = 100 μm. (e) Representative karyotypic analysis of the Eβ-iPSC2 cells at passage 19 shows normal karyotype (46, XY) (TIFF 9760 kb

  • One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    'Springer Science and Business Media LLC', 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. Results The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future

Ponthip Potirat - One of the best experts on this subject based on the ideXlab platform.

  • one step genetic correction of Hemoglobin e beta Thalassemia patient derived ipscs by the crispr cas9 system
    Stem Cell Research & Therapy, 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak Upratya, Surapol Issaragrisil
    Abstract:

    Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future.

  • Additional file 2: Figure S1. of One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Showing characterization of iPSCs derived from skin fibroblasts of a patient with Hemoglobin E/beta-Thalassemia. (a) Pluripotent gene expression of wild-type human dermal fibroblasts (wt-HDFs), parental human dermal fibroblasts (Eβ-HDFs) and Eβ-iPSCs compared with hESC line Chula2.hES, analyzed by RT-PCR. (b) Immunofluorescent staining shows expression of pluripotent markers NANOG, OCT4, SSEA-4, TRA-1-60 and TRA-1-81 in the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars = 100 μm. (c) Immunofluorescent staining shows expression of lineage markers NESTIN (ectoderm), AFP (endoderm) and SMA (mesoderm) of differentiated embryoid bodies generated from the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars: for NESTIN and SMA = 100 μm; for AFP = 50 μm. (d) Hematoxylin and eosin (H&E) staining of teratomas derived from the Eβ-iPSC2 cells at 8 weeks post implantation into nude mice. Teratomas contained tissues derived from three embryonic germ layers, sebaceous tissue (ectoderm), cartilage (mesoderm) and gut-like epithelium (endoderm). Scale bars = 100 μm. (e) Representative karyotypic analysis of the Eβ-iPSC2 cells at passage 19 shows normal karyotype (46, XY) (TIFF 9760 kb

  • One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    'Springer Science and Business Media LLC', 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. Results The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future

Kongtana Trakarnsanga - One of the best experts on this subject based on the ideXlab platform.

  • one step genetic correction of Hemoglobin e beta Thalassemia patient derived ipscs by the crispr cas9 system
    Stem Cell Research & Therapy, 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak Upratya, Surapol Issaragrisil
    Abstract:

    Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future.

  • Additional file 2: Figure S1. of One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Showing characterization of iPSCs derived from skin fibroblasts of a patient with Hemoglobin E/beta-Thalassemia. (a) Pluripotent gene expression of wild-type human dermal fibroblasts (wt-HDFs), parental human dermal fibroblasts (Eβ-HDFs) and Eβ-iPSCs compared with hESC line Chula2.hES, analyzed by RT-PCR. (b) Immunofluorescent staining shows expression of pluripotent markers NANOG, OCT4, SSEA-4, TRA-1-60 and TRA-1-81 in the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars = 100 μm. (c) Immunofluorescent staining shows expression of lineage markers NESTIN (ectoderm), AFP (endoderm) and SMA (mesoderm) of differentiated embryoid bodies generated from the Eβ-iPSC1 and Eβ-iPSC2 cells. Scale bars: for NESTIN and SMA = 100 μm; for AFP = 50 μm. (d) Hematoxylin and eosin (H&E) staining of teratomas derived from the Eβ-iPSC2 cells at 8 weeks post implantation into nude mice. Teratomas contained tissues derived from three embryonic germ layers, sebaceous tissue (ectoderm), cartilage (mesoderm) and gut-like epithelium (endoderm). Scale bars = 100 μm. (e) Representative karyotypic analysis of the Eβ-iPSC2 cells at passage 19 shows normal karyotype (46, XY) (TIFF 9760 kb

  • One-step genetic correction of Hemoglobin E/beta-Thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    'Springer Science and Business Media LLC', 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for Thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta Hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for Hemoglobin E and β-Thalassemia (HbE/β-Thalassemia), the most common Thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the Hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and Hemoglobin expression. Results The Hemoglobin E mutation of HbE/β-Thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct Hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-Thalassemia in the future